Biologists commonly wish to introduce a wide range of biological material into living cells. There exists much current research directed to the genetic transformation of living cells. Conventional technologies for introducing biological material into living cells includes electroporation, direct DNA uptake mechanisms, fusion mechanisms, microinjection mechanisms, and the use of infectious agents. However, each of these techniques suffer from some practical disadvantages.
Electroporation is a method for introducing a variety of molecules into cells by subjecting them to brief high-voltage electric pulses. For a general discussion of electroporation, see Electroporation of Eukaryotes and Prokaryotes: A General Approach to the Introduction of Macromolecules into Cells, 1988, K. Shigekawa and W. J. Dower, Biotechniques, 6:742; High-voltage Electroporation of Bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA, 1988, J. C. Miller, W. J. Dower, L. S. Tomkins, Proc. Natl. Acad. Sci. USA, 85:856-860; and A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation, 1988, I. G. Powell, M. G. Achen, A. J. Hillier, B. E. Davidson, Appl. Environ. Microbiol., 54:655-660.
General limitations of electroporation include (i) the reduction in overall cell viability caused by high applied voltages, and (ii) the inability to specifically target particular cells, especially in a complex multicellular organ.
Moreover, while electroporation methods have greatly increased the efficiency of uptake of chimeric gene constructions, such methods when used with plants are limited in plants to in vitro suspension systems. Moreover, although successfully used in the transformation of monocots as well as dicots (Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation, 1985, M. Fromm, L. P. Taylor, and V. Walbot, Proc. Natl. Acad. Sci. USA, 82:5824-5828). Electroporation methods generally require the relatively laborious and time-consuming step of removing the plant cell walls.
Uptake mechanisms generally involve suspensions of single cells, and specifically when applied to plant cells, require enzymatic removal of cell wall materials. Consequently, the uptake mechanisms are time consuming and have relatively low throughput.
One technique for uptake is the enhancement of membrane permeability by use of calcium (Ca) (Calcium Dependent Bacteriophage DNA Infection, 1972, M. Mandel and A. Higa, J. Mol. Biol., 53:159-162); and temperature shock (Frozen-thawed Bacteria as Recipients of Isolated Coliphage DNA, 1972, S. Y. Dityatkin, K. V. Lisovskaya, N. N. Panzhava, B. N. Liashenk, Biochimica et Biophysica Acta, 281:319-323).
A second technique for uptake is the use of surface-binding agents such as polyethylene glycol (PEG). For a general discussion of surface-binding agent, see High Frequency Transformation of Bacillus subtilis Protoplasts by Plasmid DNA, 1972, S. Chang, and S. N. Cohen, Mol. Gen. Genet., 168:111-115; In vitro Transformation of Plant Protoplasts With Ti-plasmid DNA, 1982, F. A. Krens, L. Molendijk, G. J. Wullems, and R. A. Schilperoort, Nature, 296:72); or such as calcium phosphate (A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA, 1973, F. L. Graham, and A. J. Van Der Eb, Virology, 52:456; Transformation of Mammalian Cells with Genes from Procaryotes and Eucaryotes, 1979, M. Wigler, R. Sweet, G. K. Sim, B. Wold, A. Pellicer, E. Lacy, T. Maniatis, S. Silverstein, and R. Axel, Cell, 16:777.
A third technique for uptake is the phagocytosis of particles into a cell. Suitable particles include liposomes (Liposome-mediated Transfer of Plasmid DNA into Plant Protoplasts, 1982, H. Uchimiya, T. Ohgawara, and H. Harada, In: A. Fujiwara (ed.), Proc. 5th Intl. Cong. Plant Tissue and Cell Culture, Jap. Assoc. for Plant Tissue Culture, Tokyo, pp. 507-508); organelles (Transplantation of Chloroplasts into Protoplasts of Petunia, 1973, I. Potrykus, Z. Pflanzenphysiol., 70:364-366); or bacteria (Plant Cell Protoplasts Isolation and Development, 1972, E. C. Cocking, Ann. Rev. Plant Physiol., 23:29-50).
Fusion mechanisms incorporate new genetic material into a cell by fusing a cell membrane with the membrane of another cell, an organelle, or a liposome. As with uptake mechanisms, plant cell fusion technologies rely upon the use of in vitro suspension systems, where cells are enzymatically stripped of any cell wall material.
Fusion can be induced with electric currents, PEG, and Sendai virus particles. For a general discussion of cell fusion, see Methods Using HVJ (Sendai Virus) for Introducing Substances into Mammalian Cells, 1980, T. Uchidaz, M. Yamaizumi, E. Mekada, Y. Okada, In: Introduction of Macromolecules Into Viable Mammalian Cells, C. Baserga, G. Crose, and G. Rovera (eds.) Wistar Symposium Series, Vol. 1, A. R. Liss, Inc., NY, pp. 169-185; and H. Harris, Cell Fusion: The Dunham Lectures, 1970, Oxford University Press.
While fusion technologies can have relatively good efficiencies in terms of numbers of cells affected, the problems of cell selection can be complex. For example, in the case of cell to cell fusion the resulting cells often have elevated ploidy, which can limit their usefulness.
Another technique is a direct method for the transfer of chromosomes by microinjection. Microinjection techniques employ extremely fine, drawn out capillary tubes which can be used as syringe needles for the direct injection of biological substances into certain types of individual cells. When small cells need to be injected, very sharp capillaries, whose tips are very easily broken or clogged, are required. Moreover, very high pressures are required to cause bulk flow through capillary apertures smaller than one micron and the regulation of such bulk flow can be difficult. The entire process is rather empirical, requiring different modifications for different cell types.
For a general discussion of microinjection techniques, see Methods for Microinjection Of Human Somatic Cells in Culture, 1973, E. G. Diacumakos, In: Methods in Cell Biology, D. M. Prescott (ed.), Academic Press, NY, pp. 287-311; Microinjection of Tissue Culture Cells, 1973, M. Graessman and A. Graessman, Methods in Enzymology, 101:482-492; Integration of Foreign DNA following Microinjection of Tobacco Mesophyll Protoplasts, 1986, A. Crossway, J. V. Oakes, J. M. Irvine, B. Ward, V. C. Knauf, C. K. Shewmaker, Mol. Gen. Genet., 202:179-185; Micromanipulation Techniques in Plant Biotechnology, 1986, A. Crossway, H. Hauptli, C. M. Houck, J. M. Irvine, J. V. Oakes, L. A. Perani, Biotechniques, 4:320-334; A Detailed Procedure for the Intranuclear Microinjection of Plant Protoplasts, 1986, T. J. Reich, V. N. Iyer, B. Scobie, B. L. Miki, Can. J. Bot.; and Efficient Transformation of Alfalfa Protoplasts by the Intranuclear Microinjection of Ti Plasmids, 1986, T. J. Reich, V. N. Iyer, B. L. Miki, Bio/Technology, 4:1001-1004.
Microinjection techniques suffer from limitations in cell recovery. Direct microinjection of plant cells is further complicated by the presence of a rigid cell wall. While protoplasts lacking the cell wall can be formed, the microinjection of plant cell protoplasts is made difficult by their extreme fragility.
Thus, a disadvantage of microinjection is that it requires single cell manipulations and is, therefore, inappropriate for treating masses of cells. The process is generally very tedious and difficult. Consequently, it tends to have very low efficiency and low throughput.
In addition to the systems mentioned above, there exist several infectious agents which can deliver nucleic acids into cells. The plant pathogen Agrobacterium tumefaciens has the innate ability to transfer a portion of DNA from a Ti (Tumor-inducing) plasmid harbored therein into an infected plant cell. By inserting foreign genes into plasmids in Agrobacterium which carry certain sequences from the Ti plasmid, the bacterial transformational trait can be used to transport the foreign genes into the genome of the infected plant cells.
Of primary importance are the Agrobacterium vectors for dicot plant cells (Genetic Transformation in Higher Plants, 1986, R. T. Fraley, S. G. Rogers, and R. B. Horsch, CRC Crit. Rev. Plant Sci., 4:1-46); and the retroviral vectors for animal cells (Prospects for Gene Therapy, 1984, F. W. Anderson, Science, 226:401-409).
Retroviruses (RNA viruses) can be used to deliver genes into animal cells. When the virus enters the cells its RNA acts as a template for reverse transcription of complementary DNA which will integrate into the genome of the host cell. This DNA can be isolated and inserted into a plasmid. This plasmid, with additional genes added, can be used to transform cells with the aid of helper retroviruses.
However, these systems are frequently difficult to control. The problem with using infectious agents such as DNA delivery systems is several-fold. First, infectious agents have limited host ranges. The mediation can only be done on an individual cellular level, typically with somatic tissues, which then must be regenerated artificially into a whole plant. This limits the applicability of Agrobacterium-mediated genetic transformation to those crop species which can readily be regenerated from types of tissues which are susceptible to Agrobacterium infection; the natural host range of Agrobacterium includes only dicotyledonous plants and a limited number of monocot species of the Liliaceae family. Likewise, retroviruses, and the expression of the DNA that they deliver, tend to be host and tissue specific.
Second, infectious agents add an additional level of complexity to the delivery process by introducing a second living system with all its concomitant complications. For example, Agrobacterium-mediated transformations may generate somoclonal variants, which spontaneously arise in plant tissues in tissue culture and which may complicate identification of transformants. In addition, infectious agents such as retroviruses are potentially dangerous--they may harm the organism being modified, or they may lead, through recombination, to the evolution of new pathogens.
Relatively recently, Sanford et al. developed a method whereby substances can be delivered into cells of intact tissues via a particle bombardment process. (High-velocity Microprojectiles for Delivering Nucleic Acids into Living Cells, 1987, T. M. Klein, E. D. Wolf, R. Wu and J. C. Sanford, Nature, 337; and Delivery of Substances into Cells and Tissues using a Particle Bombardment Process, J. C. Sanford, T. M. Klein, E. D. Wolf, and N. Allen, Particular Sci. and Technol., 5:27-37). These references teach that small, high-density, tungsten particles (microprojectiles) may be accelerated to high velocity by a particle gun apparatus. An appealing feature of the particle bombardment process is that, compared to prior art devices, it allows for the treatment of relatively many cells at once.
Sanford et al. teach that they have accelerated microprojectiles to sufficient velocities to allow plant cell penetration via the following embodiments (1) a macroprojectile (plastic bullet) and stopping plate, (2) a transferred mechanical pulse, (3) a gas (e.g., air) discharge, and (4) a centripetal acceleration system.
While the particle gun apparatus represents a proposed advance in the art, its various embodiments of the particle bombardment process have certain deficiencies. For example, in each of the various embodiments, the tungsten load suspension amount is not easily reproducible; and it is a rather laborious procedure to inoculate cultures repeatedly. Additionally, in the macroprojectile embodiment (1) and the gas discharge embodiment (3), the velocity must vary with inevitable differences in the firing characteristics; and the suspension velocity is not directly measured. Finally, the macroprojectile embodiment (3) has the following additional differences: the plastic bullet's inertia impairs velocity--making a powerful explosion necessary; the velocities are not easily changed or controlled; and the combustion gases from the gunpowder could be problematical.
Therefore, the development of a technique that can efficiently deliver noncellular biological material directly into living cells and tissues would be beneficial.
It is thus an object of the present invention to provide an apparatus and method for the acceleration of propellable matter.
It is thus another object of the present invention to provide an efficient and practical method and apparatus for inserting a wide range of noncellular biological material directly into living cells and tissues.